SnapShot: receptor dynamics at plastic synapses.

The dynamic synapse is represented by the constant mobility and exchange of components at both the cell surface and at intracellular sites. This includes thermally powered Brownian diffusion movement, followed by reversible trapping through receptor-scaffold interactions and active transport of cargo vesicles through cytoskeletal motors.

Quantifying postsynaptic receptor dynamics: insights into synaptic function.

The molecular composition of presynaptic and postsynaptic neuronal terminals is dynamic, and yet long-term stabilizations in postsynaptic responses are necessary for synaptic development and long-term plasticity. The need to reconcile these concepts is further complicated by learning- and memory-related plastic changes in the molecular make-up of synapses. Advances in single-particle tracking mean that we can now quantify the number and diffusive properties of specific synaptic molecules, while statistical thermodynamics provides a framework to analyse these molecular fluctuations. In this Review, we discuss the use of these approaches to gain quantitative descriptions of the processes underlying the turnover, long-term stability and plasticity of postsynaptic receptors and show how these can help us to understand the balance between local molecular turnover and synaptic structural identity and integrity.

Microglial TNFα orchestrates protein phosphorylation in the cortex during the sleep period and controls homeostatic sleep.

Sleep intensity is adjusted by the length of previous awake time, and under tight homeostatic control by protein phosphorylation. Here, we establish microglia as a new cellular component of the sleep homeostasis circuit. Using quantitative phosphoproteomics of the mouse frontal cortex, we demonstrate that microglia-specific deletion of TNFα perturbs thousands of phosphorylation sites during the sleep period. Substrates of microglial TNFα comprise sleep-related kinases such as MAPKs and MARKs, and numerous synaptic proteins, including a subset whose phosphorylation status encodes sleep need and determines sleep duration. As a result, microglial TNFα loss attenuates the build-up of sleep need, as measured by electroencephalogram slow-wave activity and prevents immediate compensation for loss of sleep. Our data suggest that microglia control sleep homeostasis by releasing TNFα which acts on neuronal circuitry through dynamic control of phosphorylation.

Unique properties of dually innervated dendritic spines in pyramidal neurons of the somatosensory cortex uncovered by 3D correlative light and electron microscopy.

Pyramidal neurons (PNs) are covered by thousands of dendritic spines receiving excitatory synaptic inputs. The ultrastructure of dendritic spines shapes signal compartmentalization, but ultrastructural diversity is rarely taken into account in computational models of synaptic integration. Here, we developed a 3D correlative light-electron microscopy (3D-CLEM) approach allowing the analysis of specific populations of synapses in genetically defined neuronal types in intact brain circuits. We used it to reconstruct segments of basal dendrites of layer 2/3 PNs of adult mouse somatosensory cortex and quantify spine ultrastructural diversity. We found that 10% of spines were dually innervated and 38% of inhibitory synapses localized to spines. Using our morphometric data to constrain a model of synaptic signal compartmentalization, we assessed the impact of spinous versus dendritic shaft inhibition. Our results indicate that spinous inhibition is locally more efficient than shaft inhibition and that it can decouple voltage and calcium signaling, potentially impacting synaptic plasticity.

NMDARs in granule cells contribute to parallel fiber-Purkinje cell synaptic plasticity and motor learning.

Long-term synaptic plasticity is believed to be the cellular substrate of learning and memory. Synaptic plasticity rules are defined by the specific complement of receptors at the synapse and the associated downstream signaling mechanisms. In young rodents, at the cerebellar synapse between granule cells (GC) and Purkinje cells (PC), bidirectional plasticity is shaped by the balance between transcellular nitric oxide (NO) driven by presynaptic N-methyl-D-aspartate receptor (NMDAR) activation and postsynaptic calcium dynamics. However, the role and the location of NMDAR activation in these pathways is still debated in mature animals. Here, we show in adult rodents that NMDARs are present and functional in presynaptic terminals where their activation triggers NO signaling. In addition, we find that selective genetic deletion of presynaptic, but not postsynaptic, NMDARs prevents synaptic plasticity at parallel fiber-PC (PF-PC) synapses. Consistent with this finding, the selective deletion of GC NMDARs affects adaptation of the vestibulo-ocular reflex. Thus, NMDARs presynaptic to PCs are required for bidirectional synaptic plasticity and cerebellar motor learning.

Clustering of Tau fibrils impairs the synaptic composition of α3-Na+/K+-ATPase and AMPA receptors.

Tau assemblies have prion-like properties: they propagate from one neuron to another and amplify by seeding the aggregation of endogenous Tau. Although key in prion-like propagation, the binding of exogenous Tau assemblies to the plasma membrane of naïve neurons is not understood. We report that fibrillar Tau forms clusters at the plasma membrane following lateral diffusion. We found that the fibrils interact with the Na+/K+-ATPase (NKA) and AMPA receptors. The consequence of the clustering is a reduction in the amount of α3-NKA and an increase in the amount of GluA2-AMPA receptor at synapses. Furthermore, fibrillar Tau destabilizes functional NKA complexes. Tau and α-synuclein aggregates often co-exist in patients' brains. We now show evidences for cross-talk between these pathogenic aggregates with α-synuclein fibrils dramatically enhancing fibrillar Tau clustering and synaptic localization. Our results suggest that fibrillar α-synuclein and Tau cross-talk at the plasma membrane imbalance neuronal homeostasis.

Differential regulation of glycinergic and GABAergic nanocolumns at mixed inhibitory synapses.

Super-resolution imaging has revealed that key synaptic proteins are dynamically organized within sub-synaptic domains (SSDs). To examine how different inhibitory receptors are regulated, we carried out dual-color direct stochastic optical reconstruction microscopy (dSTORM) of GlyRs and GABAA Rs at mixed inhibitory synapses in spinal cord neurons. We show that endogenous GlyRs and GABAA Rs as well as their common scaffold protein gephyrin form SSDs that align with pre-synaptic RIM1/2, thus creating trans-synaptic nanocolumns. Strikingly, GlyRs and GABAA Rs occupy different sub-synaptic spaces, exhibiting only a partial overlap at mixed inhibitory synapses. When network activity is increased by 4-aminopyridine treatment, the GABAA R copy numbers and the number of GABAA R SSDs are reduced, while GlyRs remain largely unchanged. This differential regulation is likely the result of changes in gephyrin phosphorylation that preferentially occurs outside of SSDs. The activity-dependent regulation of GABAA Rs versus GlyRs suggests that different signaling pathways control the receptors' sub-synaptic clustering. Taken together, our data reinforce the notion that the precise sub-synaptic organization of GlyRs, GABAA Rs, and gephyrin has functional consequences for the plasticity of mixed inhibitory synapses.

Reciprocal stabilization of glycine receptors and gephyrin scaffold proteins at inhibitory synapses.

Postsynaptic scaffold proteins immobilize neurotransmitter receptors in the synaptic membrane opposite to presynaptic vesicle release sites, thus ensuring efficient synaptic transmission. At inhibitory synapses in the spinal cord, the main scaffold protein gephyrin assembles in dense molecule clusters that provide binding sites for glycine receptors (GlyRs). Gephyrin and GlyRs can also interact outside of synapses, where they form receptor-scaffold complexes. Although several models for the formation of postsynaptic scaffold domains in the presence of receptor-scaffold interactions have been advanced, a clear picture of the coupled dynamics of receptors and scaffold proteins at synapses is lacking. To characterize the GlyR and gephyrin dynamics at inhibitory synapses, we performed fluorescence time-lapse imaging after photoconversion to directly visualize the exchange kinetics of recombinant Dendra2-gephyrin in cultured spinal cord neurons. Immuno-immobilization of endogenous GlyRs with specific antibodies abolished their lateral diffusion in the plasma membrane, as judged by the lack of fluorescence recovery after photobleaching. Moreover, the cross-linking of GlyRs significantly reduced the exchange of Dendra2-gephyrin compared with control conditions, suggesting that the kinetics of the synaptic gephyrin pool is strongly dependent on GlyR-gephyrin interactions. We did not observe any change in the total synaptic gephyrin levels after GlyR cross-linking, however, indicating that the number of gephyrin molecules at synapses is not primarily dependent on the exchange of GlyR-gephyrin complexes. We further show that our experimental data can be quantitatively accounted for by a model of receptor-scaffold dynamics that includes a tightly interacting receptor-scaffold domain, as well as more loosely bound receptor and scaffold populations that exchange with extrasynaptic pools. The model can make predictions for single-molecule data such as typical dwell times of synaptic proteins. Taken together, our data demonstrate the reciprocal stabilization of GlyRs and gephyrin at inhibitory synapses and provide a quantitative understanding of their dynamic organization.

Differential Membrane Binding and Seeding of Distinct α-Synuclein Fibrillar Polymorphs.

The aggregation of the protein α-synuclein (α-Syn) leads to different synucleinopathies. We recently showed that structurally distinct fibrillar α-Syn polymorphs trigger either Parkinson's disease or multiple system atrophy hallmarks in vivo. Here, we establish a structural-molecular basis for these observations. We show that distinct fibrillar α-Syn polymorphs bind to and cluster differentially at the plasma membrane in both primary neuronal cultures and organotypic hippocampal slice cultures from wild-type mice. We demonstrate a polymorph-dependent and concentration-dependent seeding. We show a polymorph-dependent differential synaptic redistribution of α3-Na+/K+-ATPase, GluA2 subunit containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and GluN2B-subunit containing N-methyl-D-aspartate receptors, but not GluA1 subunit containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and metabotropic glutamate receptor 5 receptors. We also demonstrate polymorph-dependent alteration in neuronal network activity upon seeded aggregation of α-Syn. Our findings bring new, to our knowledge, insight into how distinct α-Syn polymorphs differentially bind to and seed monomeric α-Syn aggregation within neurons, thus affecting neuronal homeostasis through the redistribution of synaptic proteins.

Increased Confinement and Polydispersity of STIM1 and Orai1 after Ca2+ Store Depletion.

STIM1 (a Ca2+ sensor in the endoplasmic reticulum (ER) membrane) and Orai1 (a pore-forming subunit of the Ca2+-release-activated calcium channel in the plasma membrane) diffuse in the ER membrane and plasma membrane, respectively. Upon depletion of Ca2+ stores in the ER, STIM1 translocates to the ER-plasma membrane junction and binds Orai1 to trigger store-operated Ca2+ entry. However, the motion of STIM1 and Orai1 during this process and its roles to Ca2+ entry is poorly understood. Here, we report real-time tracking of single STIM1 and Orai1 particles in the ER membrane and plasma membrane in living cells before and after Ca2+ store depletion. We found that the motion of single STIM1 and Orai1 particles exhibits anomalous diffusion both before and after store depletion, and their mobility-measured by the radius of gyration of the trajectories, mean-square displacement, and generalized diffusion coefficient-decreases drastically after store depletion. We also found that the measured displacement distribution is non-Gaussian, and the non-Gaussian parameter drastically increases after store depletion. Detailed analyses and simulations revealed that single STIM1 and Orai1 particles are confined in the compartmentalized membrane both before and after store depletion, and the changes in the motion after store depletion are explained by increased confinement and polydispersity of STIM1-Orai1 complexes formed at the ER-plasma membrane junctions. Further simulations showed that this increase in the confinement and polydispersity after store depletion localizes a rapid increase of Ca2+ influx, which can facilitate the rapid activation of local Ca2+ signaling pathways and the efficient replenishing of Ca2+ store in the ER in store-operated Ca2+ entry.

Estradiol modulates the efficacy of synaptic inhibition by decreasing the dwell time of GABAA receptors at inhibitory synapses.

Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c aA receptors (GABAARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABAARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABAARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABAARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABAARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABAARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.

α-synuclein assemblies sequester neuronal α3-Na+/K+-ATPase and impair Na+ gradient.

Extracellular α-synuclein (α-syn) assemblies can be up-taken by neurons; however, their interaction with the plasma membrane and proteins has not been studied specifically. Here we demonstrate that α-syn assemblies form clusters within the plasma membrane of neurons. Using a proteomic-based approach, we identify the α3-subunit of Na+/K+-ATPase (NKA) as a cell surface partner of α-syn assemblies. The interaction strength depended on the state of α-syn, fibrils being the strongest, oligomers weak, and monomers none. Mutations within the neuron-specific α3-subunit are linked to rapid-onset dystonia Parkinsonism (RDP) and alternating hemiplegia of childhood (AHC). We show that freely diffusing α3-NKA are trapped within α-syn clusters resulting in α3-NKA redistribution and formation of larger nanoclusters. This creates regions within the plasma membrane with reduced local densities of α3-NKA, thereby decreasing the efficiency of Na+ extrusion following stimulus. Thus, interactions of α3-NKA with extracellular α-syn assemblies reduce its pumping activity as its mutations in RDP/AHC.

Inhibition of group-I metabotropic glutamate receptors protects against prion toxicity.

Prion infections cause inexorable, progressive neurological dysfunction and neurodegeneration. Expression of the cellular prion protein PrPC is required for toxicity, suggesting the existence of deleterious PrPC-dependent signaling cascades. Because group-I metabotropic glutamate receptors (mGluR1 and mGluR5) can form complexes with the cellular prion protein (PrPC), we investigated the impact of mGluR1 and mGluR5 inhibition on prion toxicity ex vivo and in vivo. We found that pharmacological inhibition of mGluR1 and mGluR5 antagonized dose-dependently the neurotoxicity triggered by prion infection and by prion-mimetic anti-PrPC antibodies in organotypic brain slices. Prion-mimetic antibodies increased mGluR5 clustering around dendritic spines, mimicking the toxicity of Aß oligomers. Oral treatment with the mGluR5 inhibitor, MPEP, delayed the onset of motor deficits and moderately prolonged survival of prion-infected mice. Although group-I mGluR inhibition was not curative, these results suggest that it may alleviate the neurological dysfunctions induced by prion diseases.

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo.

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Evidence for altered dendritic spine compartmentalization in Alzheimer's disease and functional effects in a mouse model.

Alzheimer's disease (AD) is associated with a progressive loss of synapses and neurons. Studies in animal models indicate that morphological alterations of dendritic spines precede synapse loss, increasing the proportion of large and short ("stubby") spines. Whether similar alterations occur in human patients, and what their functional consequences could be, is not known. We analyzed biopsies from AD patients and APP x presenilin 1 knock-in mice that were previously shown to present a loss of pyramidal neurons in the CA1 area of the hippocampus. We observed that the proportion of stubby spines and the width of spine necks are inversely correlated with synapse density in frontal cortical biopsies from non-AD and AD patients. In mice, the reduction in the density of synapses in the stratum radiatum was preceded by an alteration of spine morphology, with a reduction of their length and an enlargement of their neck. Serial sectioning examined with electron microscopy allowed us to precisely measure spine parameters. Mathematical modeling indicated that the shortening and widening of the necks should alter the electrical compartmentalization of the spines, leading to reduced postsynaptic potentials in spine heads, but not in soma. Accordingly, there was no alteration in basal synaptic transmission, but long-term potentiation and spatial memory were impaired. These results indicate that an alteration of spine morphology could be involved in the early cognitive deficits associated with AD.

An aggregation-removal model for the formation and size determination of post-synaptic scaffold domains.

The formation and stability of synapses are key questions in neuroscience. Post-synaptic domains have been classically conceived as resulting from local insertion and turnover of proteins at the synapse. However, insertion is likely to occur outside the post-synaptic domains and advances in single-molecule imaging have shown that proteins diffuse in the plane of the membrane prior to their accumulation at synapses. We quantitatively investigated this scenario using computer simulations and mathematical analysis, taking for definiteness the specific case of inhibitory synapse components, i.e., the glycine receptor (GlyR) and the associated gephyrin scaffolding protein. The observed domain sizes of scaffold clusters can be explained by a dynamic balance between the aggregation of gephyrin proteins diffusing while bound to GlyR and their turnover at the neuron membrane. We also predict the existence of extrasynaptic clusters with a characteristic size distribution that significantly contribute to the size fluctuations of synaptic domains. New super-resolution data for gephyrin proteins established the existence of extrasynaptic clusters the sizes of which are consistent with the model predictions in a range of model parameters. At a general level, our results highlight aggregation with removal as a non-equilibrium phase separation which produces structures of tunable size.

A Simple and Powerful Analysis of Lateral Subdiffusion Using Single Particle Tracking.

In biological membranes, many factors such as cytoskeleton, lipid composition, crowding, and molecular interactions deviate lateral diffusion from the expected random walks. These factors have different effects on diffusion but act simultaneously, so the observed diffusion is a complex mixture of diffusive behaviors (directed, Brownian, anomalous, or confined). Therefore, commonly used approaches to quantify diffusion based on averaging of the displacements such as the mean square displacement, are not adapted to the analysis of this heterogeneity. We introduce a parameter-the packing coefficient Pc, which gives an estimate of the degree of free movement that a molecule displays in a period of time independently of its global diffusivity. Applying this approach to two different situations (diffusion of a lipid probe and trapping of receptors at synapses), we show that Pc detected and localized temporary changes of diffusive behavior both in time and in space. More importantly, it allowed the detection of periods with very high confinement as well as their frequency and duration, and thus it can be used to calculate the effective kon and koff of scaffolding interactions such as those that immobilize receptors at synapses.

A Primer on the Bayesian Approach to High-Density Single-Molecule Trajectories Analysis.

Tracking single molecules in living cells provides invaluable information on their environment and on the interactions that underlie their motion. New experimental techniques now permit the recording of large amounts of individual trajectories, enabling the implementation of advanced statistical tools for data analysis. In this primer, we present a Bayesian approach toward treating these data, and we discuss how it can be fruitfully employed to infer physical and biochemical parameters from single-molecule trajectories.

From the stochasticity of molecular processes to the variability of synaptic transmission.

The variability of the postsynaptic response following a single action potential arises from two sources: the neurotransmitter release is probabilistic, and the postsynaptic response to neurotransmitter release has variable timing and amplitude. At individual synapses, the number of molecules of a given type that are involved in these processes is small enough that the stochastic (random) properties of molecular events cannot be neglected. How the stochasticity of molecular processes contributes to the variability of synaptic transmission, its sensitivity and its robustness to molecular fluctuations has important implications for our understanding of the mechanistic basis of synaptic transmission and of synaptic plasticity.